“Carbon gain vs. water saving, growth vs. defence”: Two dilemmas with soluble phenolics as a joker

Plant Science 227 (2014) 21–27

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Plant Science journal homepage: www.elsevier.com/locate/plantsci

Review

“Carbon gain vs. water saving, growth vs. defence”: Two dilemmas with soluble phenolics as a joker George Karabourniotis ∗ , Georgios Liakopoulos, Dimosthenis Nikolopoulos, Panagiota Bresta, Vassiliki Stavroulaki, Sally Sumbele 1 Laboratory of Plant Physiology, Department of Crop Science, Agricultural University of Athens, Iera Odos 75, Votanikos, 11855 Athens, Greece

a r t i c l e

i n f o

Article history: Received 3 February 2014 Received in revised form 30 May 2014 Accepted 17 June 2014 Available online 23 June 2014 Keywords: Herbivores Pathogens Phenolics Photodamage Photosynthesis Tannins

a b s t r a c t Despite that phenolics are considered as a major weapon against herbivores and pathogens, the primal reason for their evolution may have been the imperative necessity for their UV-absorbing and antioxidant properties in order for plants to compensate for the adverse terrestrial conditions. In dry climates the choice concerning the first dilemma (carbon gain vs. water saving) needs the appropriate structural and metabolic modulations, which protect against stresses such as high UV and visible radiation or drought, but reduce photosynthesis and increase oxidative pressure. Thus, when water saving is chosen, priority is given to protection (including phenolic synthesis), instead of carbon gain and hence growth. At the global level, the different choices by the individual species are expressed by an interspecific negative relationship between total phenolics and photosynthesis. On the other hand, the accumulation of phenolics in water saving plants offers additional defensive functions because these multifunctional compounds can also act as pro-oxidant, antifeeding or toxic factors. Therefore phenolics, as biochemical jokers, can give the answer to both dilemmas: water saving involves high concentrations of phenolics which also offer high level of defence. © 2014 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2.

3. 4. 5. 6. 7.

Phenolics act as multifunctional secondary metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tissue localization and function of phenolics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Phenolics in superficial structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Epidermal phenolics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Mesophyll phenolics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolutionary linkage between phenolics and terrestrial abiotic stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protection demand is negatively related to photosynthetic capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Accumulation of leaf phenolics relates to the “carbon gain vs. water saving” dilemma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phenolics, as biochemical jokers, also answer to the dilemma “growth vs. defence” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks: the hierarchy of plant dilemmas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

21 22 23 23 23 24 25 25 26 26 26 26

1. Phenolics act as multifunctional secondary metabolites ∗ Corresponding author. Tel.: +30 2105294286. E-mail address: [email protected] (G. Karabourniotis). 1 Present address: Institute of Agricultural Research for Development, Yaounde, Cameroon. http://dx.doi.org/10.1016/j.plantsci.2014.06.014 0168-9452/© 2014 Elsevier Ireland Ltd. All rights reserved.

It has been proposed that resource allocation to secondary metabolism is antagonistic to that of primary metabolism. In other words plants have to cope with the dilemma of

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Table 1 Tissue localization, spectral, chemical and bioactivity properties of major classes of phenolic compounds related to their protective and defensive roles in plants. Phenolic class

Tissue localization

max (nm)

ε in max (×104 M−1 cm−1 )a

Prooxidant activityb

ROS scavenging capacityc

Toxicity to herbivores and pathogensd

Hydroxybenzoates and hydroxycinnamates

Cuticle, epidermis, mesophyll

227–332 (UV-C, UV-B, UV-A)

1.8–1.9

215–469

2.03 ± 0.67

Low to medium

Flavonol aglycones Flavonol glycosides

Superficial structures Epidermis, mesophyll

250–390 (UV-C, UV-B, UV-A)

1.5–2.1

469–1841

2.92 ± 1.39 1.97 ± 0.72

High Low to medium

Flavone aglycones Flavone glycosides

Superficial structures Epidermis, mesophyll

250–350 (UV-C, UV-B, UV-A)

0.8–2.1

69–1745

1.66 ± 0.38 1.27 ± 0.67

High Low to medium

Flavanonols

Superficial structures

290–340 (UV-B, UV-A)

No data

–

1.65 ± 0.36

No data

Flavanone aglycones Flavanone glycosides

Superficial structures Epidermis, mesophyll

225–330 (UV-B, UV-A)

1.8–2.3

44–430

1.58 ± 0.20 0.94 ± 0.20

High Low to medium

Catechins

Mainly mesophyll

270–280 (UV-C, UV-B)

ca. 0.4

–

3.68 ± 1.20

High

Tannins

Mainly mesophyll

Depending on structure

Depending on structure

Depending on structure

Highe

High

Anthocyanins

Epidermis, mesophyll

267–275 (UV-C, UV-B), 475–545 (Vis)

Low to medium in UV depending on acylation

2.89 ± 1.45 2.55 ± 0.63

Lowf

a

Data from [71,72]. Measured as the cooxidation rate of ascorbate (k × 103 min−1 ); data from [36] c Measured as Trolox equivalent antioxidant capacity; the concentration of Trolox with the equivalent antioxidant capacity of a 1 mM concentration of the experimental substance. Data from [45,64,73]. Values are means ± SD. d Data from [5] e Trolox equivalents of 2.6 or considerably higher have been reported depending on structure and degree of polymerization [74]. f Anthocyanins display low toxicity but possess other indirect defensive roles through colouration. b

investing the photosynthetic products between the conflicting demands of growth (including maintenance cost) and defence [1]. However, in many cases, resource allocation to secondary metabolism is not seriously compromised by leaf or shoot growth [2], and usually reflects the need of protection of primary metabolic processes against the side effects of abiotic stress factors. A coordinated regulation of primary and secondary metabolism frequently leads to parallel and not reciprocal changes of the corresponding metabolites [3]. Thus, in many cases, resource allocation to secondary metabolism is not exclusively determined by the demands of defence against biotic stresses, but also by the protection needs against abiotic ones. This may be a result of the multifunctionality of almost all classes of secondary metabolites. Among them, phenolics are considered as fulfilling the wider array of functions. Phenolics are the most commonly studied compounds because of their universal presence in high concentrations (requiring significant resources) and their significant roles in plant cells and tissues [4,5]. The term “phenolic” is used to define carbon-based metabolites that possess one (simple phenols) or more (polyphenols) hydroxyl substituents bonded onto an aromatic ring. These compounds are considered to be among the most important chemical weapons against a diverse array of herbivores ([1] and the literature therein). However, this highly diverse group of secondary metabolites fulfils multiple functions: (a) as constitutive bioactive compounds, they take part in the defence against herbivores or pathogens. Phenolics may also be synthesized de novo during in situ defence responses which include the accumulation of phytoalexins or during hypersensitive response, a systemic plant reaction against pathogens. Induced synthesis of phenolics is not examined in this review. Notably, the constitutive accumulation of phenolics is related to the growth vs. defence dilemma. Effective defence requires considerable amounts of carbon skeletons and energy for the synthesis of secondary metabolites (among which phenolics predominate). Therefore, effective defence usually retards the investment of carbon and energy in growth processes. For this reason, highly defended plants usually show low growth rates [1], (b) as absorbing filters, they reduce the penetration of UV and visible radiation into sensitive targets [6], (c) as antioxidants, they reduce

the damage caused by reactive oxygen species (ROS) [7,8], (d) as regulators of soil processes they control the recycling and thus the availability of nutrients for plants and soil microbes [9] and (e) as signal molecules they play a significant role in the interactions between plants and other organisms [10], as well as in morphogenesis [11]. It should be also noted that function (e) requires the presence of phenolics in considerably lower concentrations than each of the other functions. Phenolics are a group of compounds with numerous separate chemical structures. Thus, two questions should be addressed: (i) Is the multifunctionality of phenolic compounds a result of the different chemical properties of each individual subclass or could the majority of phenolic compounds be involved in more than one (or even in all) of the above different functions? And (ii) Is there a hierarchy among these functions that could affect plant survival? Concerning the first question, data from Table 1 show that all phenolic subclasses show similar spectral and biochemical properties, differing much less that an order of magnitude between different structures. Concerning their in vivo protective role, our data show that total phenolics, but also the condensed tannins subpool, are similarly correlated with photosynthetic capacity. This indicates that at least one phenolic subpool shows similar protective behaviour to that of the total pool (see Fig. 1, Table 1 and Section 2). Concerning the second question, the antioxidant and UV protective function of phenolics probably has priority over defence against biotic stress factors (see Sections 3 and 5), but both their protective and defensive roles may occur in parallel (see Section 6).

2. Tissue localization and function of phenolics The diverse group of phenolics is subdivided at the molecular level into many sub-groups, such as simple phenols, lignans, coumarins, flavonoids, tannins, quinones, etc., based on the construction of the carbon skeleton, the kind of substituent and the degree of polymerization [4]. Moreover the solubility and the toxicity of each molecule depend on glycosylation, whereas their antioxidant properties depend on the number of hydroxyl

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Fig. 1. Fit of reciprocal model and regression coefficient for net photosynthetic capacity per mass (Amax,m ) vs. concentration of phenolic compounds per mass (Phm ); r = 0.64, at p < 0.01. Inset: fit of reciprocal model and regression coefficient for net photosynthetic capacity per mass (Amax,m ) vs. concentration of condensed tannins per mass (CTm ); r = 0.50, at p < 0.001. Data obtained from [47,70]. Data concerning Japanese species were kindly provided by Prof. Ishida. Data concerning the three genotypes of wheat and the three of barley (crop plants) obtained from P. Bresta and V. Stavroulaki (unpublished results). Indexes for plant life form and origin are common for the two graphs.

groups. The quantitative determination of phenolics is usually based on a proximate assay technique (usually the Folin–Ciocalteu assay), which is based on the quantification of the total concentration of phenolic hydroxyl groups, irrespectively of the particular molecules present in the extracts. The concentration of other interfering antioxidants (such as ascorbate) is usually significantly lower than that of phenolics [4]. Thus, the method provides a surrogate metric for the antioxidant capacity of the soluble phenolic pool. However this simplified approach does not provide detailed information because the phenolic pool of plant tissues consists of a great number of separate chemical structures and these molecules are usually unevenly distributed within different tissues [12]. Therefore, “total phenolics” flattens all spatial heterogeneities in the distribution of specific classes of compounds and their concentration in plant tissues. A significant portion of the non-extractable phenolics is covalently bound to the cutin or cell wall polymers via ether or ester bonds and thus not included in the measurement of “total phenolics” [13,14]. Consequently, a question arises: which subclasses of phenolics are determined and which functions do they serve? As it is detailed below (Sections 2.1–2.3), although the composition of phenolic compounds depends on their localization on a tissue, cell and subcellular level, many subclasses of phenolic compounds (e.g. flavonoids) are widely distributed in the leaf. It is obvious that these compounds possess the same properties regardless of their localization. However, the contribution of each molecule in specific functions (e.g. UV-screening or antioxidant protection), changes depending on the particular environment (e.g. epidermal vs. mesophyll or valuolar vs. chloroplastic).

metabolites. In some plant species high levels of exudates are produced in glandular hairs and form a continuous layer on the leaf surface ([17], see also Fig. 2). The exudates usually form a complex, resinous mixture of secondary metabolites, consisting mainly of terpenoids, flavonoid aglycones and phenolic acids, frequently imbedded in a lipophilic matrix [17]. Usually, the composition of superficial phenolics differs from that of the internal pool, i.e. mesophyll and epidermal cell phenolics [14]. Superficial phenolics are usually only a minor fraction of the total leaf pool [14]. The presence of phenolics in the cuticle, the nonglandular hairs and the exudates of glandular hairs are responsible for the UV-B filtering capacity of these superficial structures [18,19], supplying additional protection against UV-B radiation. Stomatal guard cells have a thick cuticular layer covering containing high concentration of wax-bound phenolics that provides a special protection against UV radiation [20]. The phenolic content of these superficial tissues provides also the first line of defence against pathogens and insects due to the hydrophobicity of the phenolic aglycones (see Table 1). 2.2. Epidermal phenolics Epidermal cells accumulate a variety of different soluble phenolics in their vacuoles, such as glycosylated flavonoids (mainly quercetin and kaempferol derivatives), hydroxycinnamic acids [21], anthocyanins [22], and in some cases tannins [23] (Fig. 2). These phenolics are responsible for the strong UV absorbing capacity of the epidermis. UV-B stress results in the production of ROS [24] and epidermal phenolics may also act as antioxidants that reduce the oxidative damage caused by UV radiation [25].

2.1. Phenolics in superficial structures 2.3. Mesophyll phenolics Some phenolics (mainly ferulic and p-coumaric acid, flavonoid glycosides and aglycones) are located in the cuticle or in epidermal appendances, glandular or non-glandular hairs [13]. Non-glandular hairs often create dense layers covering the upper or lower leaf surface. These hairs contain extractable phenolics, particularly flavonoids, associated with cell walls [15] and, in some cases, tannins [16]. Glandular hairs produce and store mixtures of secondary

Mesophyll cells also accumulate different groups of phenolics, including flavonoid glycosides, hydroxycinnamic acids, anthocyanins and tannins [26,27] (Fig. 2). The accumulation of phenolics varies among different types of photosynthetic cells. The phenolic content of palisade cells is usually higher than that of spongy cells [28]. The epidermis absorbs a considerable portion of the UV

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Fig. 2. Schematic representation of typical anatomical features, often occurring in hypostomatic leaves, showing the localization of the different subgroups of phenolics at tissue and cellular level. The main roles of phenolics of each feature are shown in magenta. Two-headed arrow shows the hypothetical gradient of oxidative stress and hypothetical concentration of phenolic compounds across mesophyll tissues. For details, see text.

radiation. Thus, the main role of mesophyll phenolics could be related to the protection against ROS, especially under adverse conditions [29]. Phenolics in photosynthetic mesophyll cells are distributed in different subcellular compartments having different functional roles (Fig. 2). Vacuolar phenolics, the predominant group, may serve as substrates of class III peroxidases, efficiently scavenging H2 O2 [27,30]. This mechanism is thought to have a crucial role in the homeostasis of H2 O2 levels, scavenging excess H2 O2 diffusing from the chloroplast or other cellular compartments. This function may be essential under conditions of severe stress when H2 O2 production in chloroplasts and mitochondria exceeds the scavenging capacity of these organelles [30,31]. Oxidized vacuolar phenolics can act as prooxidants in defensive reactions (see Section 6), or can be regenerated by an ascorbate-mediated regeneration cycle, acting as effective antioxidants [30]. Phenolics, mainly flavonoids, are also accumulated in other sensitive cell compartments, such as the chloroplasts [31,32], and the nucleus [33] protecting them from oxidative damage. Mesophyll phenolics are also located in the apoplast, where they are substrates for lignin or suberin biosynthesis [34]. These two processes take place mainly in the cell walls of water conducting vessels and neighbouring fibres or in the cell walls of tissues of underground and aboveground plant parts, endodermis and bark respectively. They involve the formation of a three-dimensional polyphenolic matrix within the carbohydrate matrix of the cell wall offering mechanical strengthening (lignin) or waterproofing (suberin) and constitutive and/or induced defence against pathogens [34]. According to Table 1, in the majority of cases, flavonoid aglycones show higher antioxidant activity and higher toxicity compared to glycosylated molecules. This is notable since aglycones are located in superficial structures which form a first line

of defence against biotic stress factors. The decline in antioxidant activity is an inevitable consequence of glycosylation since these molecules have to be contained in the aqueous phase of the protoplast (mainly in the vacuole) although flavonoids (with a maximum antioxidant activity) are located in the chloroplast [32]. Tannins display considerable, but also very variable, prooxidant activity. The variability in activity depends on chemical structure; ellagitannins (hydrolysable tannins) show much higher oxidative activity that condensed tannins and galloyl glucosides [35]. A variety of phenolic molecules have been tested and all of them exhibit a considerable degree of prooxidant activity (Table 1). Large differences exist between individual molecules that cannot be classified based on class of compound. For instance, quercetin (a flavonol, possessing a catechol B-ring) exhibits a prooxidant activity (as capacity to cooxidize ascorbate) of 1720 k × 103 min−1 while kaempferol (also a flavonol but equipped with a phenol B-ring) shows a four-fold lower prooxidant activity [36]. All the above suggest that all plants are equipped with at least a baseline UV screening, antioxidant and prooxidant capacity while any differences should primarily depend on concentration although specific composition of phenolics in each species may also contribute. 3. Evolutionary linkage between phenolics and terrestrial abiotic stress The existing data indicate that the metabolic pathways leading to the synthesis of phenolic compounds are evolutionary linked to the conquest of land by plants and their protection against the abiotic stress factors associated with the terrestrial environment [37–39]. The transition from the relatively stable aqueous

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environment to the fluctuating gaseous terrestrial one, exposed plants to the risk of dehydration, as well as to their exposure to the harmful UV and high intensities of visible radiation [39,40]. The evolution of the cuticle (which usually contains a variable amount of phenolic compounds, see Section 2) is vital for water retention. Flavonoids and simple phenolics are also deposited mainly in specific subcellular compartments of epidermal appendances, as well as in epidermal and mesophyll cells (see Section 2). They may have been critical as UV-B absorbing filters for the protection of early land plants [4]. Compared to scytonemin and mycosporinelike aminoacids, screening pigments in some evolutionary ancient photosynthetic organisms such as cyanobacteria [41], the evolution of phenolics as carbon-based screening pigments does not require nitrogen. Thus, the evolution of phenolics resulted in both efficient protection and decrease of nitrogen demands. The gradual expansion of plants in the terrestrial environment had another significant consequence: the establishment of the present atmosphere. Intensive photosynthesis (also by marine photosynthetic organisms) and weathering caused the depletion of atmospheric CO2 and the increase in O2 concentration, reaching an O2 /CO2 ratio of above 1000 [42]. The high O2 /CO2 ratio and the resultant evolution of aerobic metabolic processes inevitably led to the increased production of ROS [42,43]. Additionally, there are indications of an increasing biochemical complexity of phenolics during plant evolution [40,44] resulting in increased antioxidant capacity of the resulting molecules [45]. Thus the high atmospheric O2 /CO2 ratio probably triggered the expansion of the metabolic paths leading to the synthesis of numerous phenolic compounds with strong antioxidant properties. It is probable that, at the evolutionary level, the interspecific variation in the concentration of phenolics may reflect different selective pressures from oxidative stress and potential risk of photodamage [1]. It seems therefore that in most cases the dilemma growth vs. defence was not the primary determinant for the evolution of phenolics biosynthetic paths [1]. 4. Protection demand is negatively related to photosynthetic capacity Evolution led to the expansion of the metabolic pathways for the synthesis of phenolic compounds that are primarily involved in the protection against photodamage caused by irradiation and oxidative stress [1]. Thus, it is expected that the interspecific concentration of these compounds in leaves should vary according to the protection demands of each species. Furthermore, the concentration of phenolics should be functionally integrated with photosynthetic capacity because stress factors that limit photosynthesis tend to increase the risk of photodamage [46]. The photosynthetic capacity and the concentration of leaf phenolics are negatively correlated [47] (see also Fig. 1 of the present review which includes additional data) indicating a probable physiological link between gas exchange properties and the phenolic pool at the interspecific level. The relationship between photosynthetic capacity and the concentration of leaf condensed tannins is similar to that of total phenolics (Fig. 1, insert). This indicates that the subpool of condensed tannins may fulfil similar protective role(s) as the total phenolic pool. However, further experimental proof is required to confirm this suggestion. 5. Accumulation of leaf phenolics relates to the “carbon gain vs. water saving” dilemma The observed negative relationship between photosynthetic capacity and the concentration of phenolics (Fig. 1) probably represents the gradient between maximum carbon gain and maximum protection (i.e. the choice of each plant species or life form (Fig. 1)

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concerning the dilemma carbon gain vs. protection (or defence)). Since total phenolics method gives a surrogate metric of the antioxidant defensive capacity of plant species, the different strategy adopted by each of the different plant species and life forms must be balanced between the above two extremes because low photosynthetic capacity results in high oxidative pressure and vice versa. The different strategy chosen by each plant species is thus reflected to the level at which phenolics accumulate. Fast growing plants, such as annuals, that complete their life cycle under favourable conditions have evolved to maximize instantaneous carbon gain which, in turn, requires sufficient water and nutrients input. In these plants, investment to defence and protection is inevitably low. Therefore, annual species tend to have a high photosynthetic capacity and low phenolic concentrations (Fig. 1). An extreme deviation of the above strategy can be seen in crop plants. The data points of wheat and barley genotypes are outliers of the reciprocal plot of Fig. 1 (not shown). The photosynthetic capacity of these plants is higher than expected based on their concentration of phenolics, probably due to genetic optimization by artificial selection and breeding programmes. Conversely, slow growing species such as perennial shrubs and trees, have low photosynthetic capacities and high phenolic concentrations (Fig. 1). This could be attributed to a long-term probability that these long-lived plant species evolved to confront both biotic and abiotic (primarily water shortage) stress factors. In perennials that are acclimated and adapted to limited resources, photosynthetic capacity keeps pace with the low nutrient and water uptake rates [48]. In these plants, high photosynthetic capacity would be a wasteful and ineffective investment. At the interspecific level, low photosynthetic capacity is related not only to biochemical restrictions (nitrogen allocation to photosynthetic apparatus, specific activity of photosynthetic enzymes, potential rates of transpiration), but also to leaf structural limitations (smaller stomata and higher stomatal density, thick cell walls of mesophyll cells, and probably high leaf mass per area) leading to a reduced mesophyll conductance [49]. Some phenolic signal molecules contribute to the development of biochemical restrictions and structural limitations of photosynthesis. Salicylic acid, which has a significant role in the induction of protective mechanisms against biotic and abiotic stress factors [50], affects photosynthetic reactions by slowing down photosystem II electron transport and by inducing stomatal closure [51]. Salicylic acid is also accumulated under water stress conditions and may be involved in the regulation of water balance and in the activation of the antioxidant system [52] Salicylic acid could contribute to maintaining cellular redox homeostasis through the regulation of PAL activity (and thus the biosynthesis of phenolics), as well as the activity of antioxidant enzymes such as polyphenol oxidases and superoxide dismutases [53]. Moreover, polyphenols (particularly colourless flavonoids and anthocyanins) act as developmental regulators/signal molecules causing morpho-anatomical adjustments of plants and plant organs by affecting both the movement and the catabolism of auxin. Flavonoids, such as quercetin, apigenin, kaempferol or other aglycones, have been shown to inhibit polar auxin transport in apical tissues [54]. Flavonoids are effective in establishing auxin gradients also by regulating auxin catabolism through their effect on auxin oxidase (either affecting positively or negatively, e.g. depending on the quercetin/kaempferol ratio). The above phenomena contribute to structural modifications such as branching architecture and thicker leaf lamina that may allow plants to confront with a wide array of abiotic stresses [31]. Moreover, abscisic acid regulates the activity of MYB transcription factors, and hence the biosynthesis of flavonoids, anthocyanins and condensed tannins [55]. The expression of the gene encoding the FaMYB10 transcription factor is increased in water-stressed fruits of strawberry, which is accompanied by an increase in both ABA

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and anthocyanin content [56]. Thus, abscisic acid offers probably a link between drought responses and phenolics. Nevertheless, leaf structural limitations restrict CO2 diffusion into the mesophyll, thus the CO2 supply to the layers of the photosynthetic parenchyma is probably insufficient [57]. CO2 restrictions are expected to be most severe in upper layers of photosynthetic parenchyma of hypostomatic leaves. Thus, photosynthesis in these cells probably functions in a high light and low CO2 microenvironment which is responsible for a higher photooxidative risk [58]. Under these conditions, the accumulation of phenolics renders sufficient protection due to the antioxidant and UV absorbing properties of these molecules (Fig. 2). Indeed, in leaves acclimated to high light intensities, antioxidant flavonoids occur to a great extent in the upper (both epidermal and palisade) cell layers [8,59]. All the above indicate that the interspecific variations in the concentration of phenolics reflect the answer to the dilemma “carbon gain vs. water saving”. Water loss from leaves is the inevitable cost of acquiring CO2 for photosynthesis. Thus, water saving plants evolved to accumulate protective compounds (by synthesizing antioxidants including phenolics) instead of diverting the respective carbon to the photosynthetic machinery to achieve maximum growth. Phenolics act in concert with other protective molecules in plant cells, including enzymatic and non-enzymatic scavengers of ROS, probably compensating for deficiencies of such molecules during periods of stress [60]. Moreover the synergistic interaction of sugars and phenolic compounds may be part of the redox system [61]. Water stress usually causes an increase in soluble carbohydrates levels and sugar signalling cascades may lead to the stimulation of the synthesis of phenolics [62].

6. Phenolics, as biochemical jokers, also answer to the dilemma “growth vs. defence” In water saving plants the carbon cost for biosynthesis of phenolics as protective compounds is considerably high. For this reason the biosynthesis of additional secondary metabolites that would fulfil the defensive demands of plants is rather prohibitive. However, the multifunctionality of phenolics (see Sections 1 and 6) enables their involvement also in defensive mechanisms based on their role mainly as prooxidants. But how phenolics can act as biochemical jokers? In intact tissues these compounds protect cells from UV and ROS damage. ROS scavenging capacity of phenolics is primarily attributed to the high reactivity of hydroxyl groups that donate hydrogen and an electron to hydroxyl, peroxyl and peroxynitrite radicals, giving rise to a relatively stable phenoxyl radical [63,64]. In undamaged tissues the phenoxyl radicals usually do not behave as harmful prooxidants because they are rapidly converted to inactive products by enzymatic or non-enzymatic reduction, as well as by polymerization reactions [65]. Upon tissue damage or in a suitable environment such as the digestive organs of a feeding insect, leaf phenolics (as phenoxyl radicals produced through oxidative reactions) can act as prooxidants creating oxidative stress within insect tissues [43,60,66,67]. Conditions prolonging the radical lifetime, such as the presence of metal ions (i.e. Fenton reactions), corroborate the prooxidant activity of the phenoxyl radicals [65]. Thus the molecular behaviour of phenolic compounds (i.e. their action either as antioxidants or as prooxidants) may depend on physiological parameters such as the ROS level, the intactness of the cell and tissues, and the occurrence of suitable enzymes and metal ions. The accumulation of phenolics is also related to the defence against herbivores and pathogens for two additional reasons: (a) leaves containing high concentrations of phenolics (including tannins) are characterized by low nutritional value [68] and (b) phenolic compounds can act as toxins directly at the site of pathogen attack, probably by enzymatic

oxidation and covalent binding of leaf quinones to some microbial proteins [69].

7. Concluding remarks: the hierarchy of plant dilemmas According to the above, the accumulation of phenolics offers considerable carbon and energy economy because the same molecules are effective in both protection and defence. By taking advantage of the multifunctionality of phenolics, plants accomplish significant economy in available resources. The evolution of phenolic compounds as protective agents against radiation also negated the need for nitrogen containing screening molecules. In water-limiting environments, high levels of phenolic compounds minimize the risk of photoinhibition and photooxidation and may offer advantages in water-saving plants compared to plants which show high carbon gain and rapid growth. This was the first crucial dilemma that plants had to answer through the evolutionary transition to the terrestrial challenging environment which made phenolic compounds an essential tool for plant survival. In other words the dilemma carbon gain vs. water saving has probably gained an evolutionary priority over the second one (growth vs. defence). Nevertheless, the accumulation of phenolics also addressed the second dilemma: leaves of plants characterized by the “water saving” mode are already equipped with phenolics and can also be characterized as profoundly defended. In order to understand the multifunctionality of phenolics, the phenomena causing the negative relationship between photosynthetic capacity and concentration of phenolics (and condensed tannins) need to be revealed. Structure-function relationships controlling the balance between carbon gain and water saving (such as the mesophyll conductance and coordinated stomatal and xylem structural modulations) may affect the leaf phenolic pool not only at the interspecific, but also at the intraspecific level. Whether the concentration of leaf phenolics is related to these parameters remains to be answered.

Acknowledgements The authors would like to thank Prof. Atsushi Ishida (Centre for Ecological Research, Kyoto University, Japan) for providing data and making useful comments on the manuscript.

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